The present invention, in some embodiments thereof, relates to a scaffold comprising a protein cross-linked by a synthetic polymer, and more particularly, but not exclusively, to methods of generating and using same in tissue engineering.
Tissue engineering, i.e., the generation of new living tissues in vitro, is widely used to replace diseased, traumatized or other unhealthy tissues. The classic tissue engineering approach utilizes living cells and a basic scaffold for cell culture. Thus, the scaffold structure attempts to mimic the natural structure of the tissue it is replacing and to provide a temporary functional support for the cells.
Tissue engineering scaffolds are fabricated from either biological materials or synthetic materials, such as polymers. Synthetic materials such as polyethylene glycol (PEG), hydroxyapatite/polycaprolactone (HA/PLC), polyglycolic acid (PGA), poly-L-lactic acid (PLLA), polymethyl methacrylate (PMMA), polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate (P4HB), polypropylene fumarate (PPF), polyethylene glycol-dimethacrylate (PEG-DMA), beta-tricalcium phosphate (beta-TCP) and polytetrafluoroethylene (PTFE) provide precise control over the mechanical properties of the material [Drury and Mooney (2003)].
Common scaffold fabrication methods are based on foams of synthetic polymers. However, cell migration into the depth of synthetic scaffolds is limited by the lack of oxygen and nutrient supply. To overcome such limitations, new approaches utilizing solid freeform fabrications and internal vascular architecture have been developed [Sachlos and Czernuszka (2003)]. Likewise, freeze-drying methods are also employed to create unique three-dimensional architectures with distinct porosity and permeability.
Scaffolds made of PEG are highly biocompatible [Merrill and Salzman (1983)] and exhibit versatile physical characteristics based on their weight percent, molecular chain length, and cross-linking density [Temenoff et al. (2002)]. In addition, PEG hydrogels are capable of a controlled liquid-to-solid transition (gelation) in the presence of cell suspension [Elbert and Hubbell (2001)]. Moreover, the PEG gelation (i.e., PEGylation) reaction can be carried out under non-toxic conditions in the presence of a photoinitiator [Elisseeff et al. (2000); Nguyen and West (2002)] or by mixing a two-part reactive solution of functionalized PEG and cross-linking constituents [Lutolf and Hubbell (2003)].
A range of tissue engineering products based on collagen scaffolds are currently under development, some of which have reached the market. For example, collagen gels seeded with fibroblasts have been used as the “dermal” layer of the artificial skin sold under the tradename APLIGRAFT (Sandoz A G, Basel, Switzerland), and collagen sponges have been used as an osteoconductive carrier of bone morphogenic protein-2 (BMP-2) for spine fusion and the treatment of long bone fractures.
Collagen based biomaterials have been formed into fibers, film, sheets, sponges and dispersions of fibrils. Many of these forms could potentially be used as tissue engineering scaffolds in the repair or augmentation of body tissue.
Collagen gels are made from a network of fibrils that exhibit poor physical strength and super-physiological tissue porosity. The specific conformation of fibrils combined with the open pore structure of the interpenetrating network leaves the protein backbone easily accessible and susceptible to freely diffusing proteases from the surrounding host tissue or cell culture system. This often results in uncontrolled and premature deterioration of the scaffold in the presence of cell-secreted proteases [Hubbell (2003); Friess (1998); Nicolas and Gagnieu (1997)]. The discrepancies in structure and proteolytic susceptibility of reconstituted protein hydrogels compared to natural tissues still leaves much to be desired from the biologic scaffold systems in many practical tissue engineering applications.
Some techniques for improving the physical properties of collagen gels are based on covalent cross-links, using aldehydes, carbodiimides, and N-hydroxysuccinimides (NHS) [Park et al. (2002); Ma et al. (2004)], for example. Many of the cross-linking procedures offer some improvements over the physical stability and reduced enzymatic susceptibility of the scaffold, but do so by introducing a cytotoxic manufacturing step which requires extensive washes and increases the likelihood that residual toxins in the scaffold will affect cellular activity [Nimni et al. (1987); Friess (1998)].
Collagen and fibrin gels can also be processed by freeze-drying to increase the tensile strength and modulus of the protein network [Schoof et al. (2001); Buttafoco et al. (2006); Pieters et al. (2002)]. Fortier and coworkers have described a single-step process for generating a scaffold comprising albumin and polyethylene glycol, having an immobilized enzyme [Jean-Francois and Fortier (1996); Jean-Francois et al. (1997); Gayet and Fortier (1995); D'Urso et al. (1995)].
WO 1995/015352 describes a hydrogel comprising albumin and bifunctionalized polyethylene oxide.
WO 2005/061018 describes a scaffold comprising a naturally occurring protein such as fibrinogen cross-linked by PEG, by attaching modified PEG molecules to cysteine residues of the protein.